Cyclic peptides for treatment for cachexia

ABSTRACT

A highly selective melanocortin-4 receptor antagonist cyclic peptide of the formula 
                         
where R 1 , R 2 , R 3 , R 4a , R 4b , R 5 , R 6 , R 7 , x, y and z are as defined in the specification, and a method of treating body weight disorders, including cachexia, sarcopenia and wasting syndrome or disease, and treating inflammation and immune disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 10/638,071, filed Aug. 8, 2003, and issued as U.S.Pat. No. 7,176,279 on Feb. 13, 2007, entitled “Cyclic PeptideCompositions and Methods for Treatment of Sexual Dysfunction”, to ShubhD. Sharma, et al., which is a continuation-in-part of InternationalApplication No. PCT/US02/22196, International Publication No. WO03/006620, filed on Jul. 11, 2002, entitled “Linear and CyclicMelanocortin Receptor-Specific Peptides”, to Shubh D. Sharma, et al.,and the specification and claims thereof of each are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

The present invention relates to cyclic peptides that arehighly-specific antagonists for the melanocortin-4 receptor (MC4-R), andwhich may be used in the treatment of a variety of body weight disordersincluding cachexia, sarcopenia and wasting syndrome or disease, and fortreatment of inflammation and immune disorders.

2. Description of Related Art

Note that the following discussion refers to a number of publications byauthor(s) and year of publication, and that due to recent publicationdates certain publications are not to be considered as prior artvis-a-vis the present invention. Discussion of such publications hereinis given for more complete background and is not to be construed as anadmission that such publications are prior art for patentabilitydetermination purposes.

Melanocortin Receptors. A family of melanocortin receptor types andsubtypes have been identified, including melanocortin-1 receptors(MC1-R) expressed on normal human melanocytes and melanoma cells,melanocortin-2 receptors (MC2-R) for ACTH (adrenocorticotropin)expressed in cells of the adrenal gland, melanocortin-3 andmelanocortin-4 receptors (MC3-R and MC4-R) expressed primarily in cellsin the hypothalamus, mid-brain and brainstem, and melanocortin-5receptors (MC5-R), expressed in a wide distribution of peripheraltissues.

Significant work has been done in determining the structure ofmelanocortin receptors, including both the nucleic acid sequencesencoding for the receptors and the amino acid sequences constituting thereceptors. MC4-R is a G protein-coupled, 7-transmembrane receptor thatis believed to be expressed primarily in the brain. Inactivation of thisreceptor by gene targeting has been reported to result in mice with thematurity-onset obesity syndrome that is associated with hyperphagia,hyperinsulinemia, and hyperglycemia (Huszar D., Lynch C. A.,Fairchild-Huntress V., et al. Targeted disruption of the melanocortin-4receptor results in obesity in mice. Cell 88:131-141 (1997)). MC4-R is amolecular target for therapeutic intervention in energy homeostasis.

In general, compounds specific for MC4-R, and secondarily compoundsspecific for MC3-R or MC5-R, are believed to be useful in regulation ofmammalian energy homeostasis, including use as agents for attenuatingfood intake and body weight gain. MC4-R antagonists are believed to beuseful for weight gain aid, such as for use in treatment of cachexia,sarcopenia, wasting syndrome or disease, and anorexia. MC4-R agonists,by contrast, are believed to be useful for decreasing food intake andbody weight gain, such as for treatment of obesity. Compounds that areantagonists specific for MC3-R and MC4-R are additionally believed to beuseful in regulating blood pressure, heart rate and otherneurophysiologic parameters.

Cachexia and Other Wasting Diseases. Body weight disorders include oneor more “wasting” disorders (e.g., wasting syndrome, cachexia,sarcopenia) which cause undesirable and unhealthy loss of weight or lossof body cell mass. In the elderly as well as in cancer and AIDSpatients, wasting diseases can result in undesired loss of body weight,including both the fat and the fat-free compartments. Wasting diseasescan be the result of inadequate intake of food and/or metabolic changesrelated to illness and/or the aging process. Cancer patients and AIDSpatients, as well as patients following extensive surgery or havingchronic infections, immunologic diseases, hyperthyroidism, Crohn'sdisease, psychogenic disease, chronic heart failure or other severetrauma, frequently suffer from wasting disease. Wasting disease issometimes also referred to as cachexia, and is generally recognized as ametabolic and, sometimes, an eating disorder. Cachexia may additionallybe characterized by hypermetabolism and hypercatabolism. Althoughcachexia and wasting disease are frequently used interchangeably torefer to wasting conditions, there is at least one body of researchwhich differentiates cachexia from wasting syndrome as a loss offat-free mass, and particularly, body cell mass (Roubenoff R. Thepathophysiology of wasting in the elderly. J. Nutr. 129(1 SSuppl.):256S-259S (1999)). Sarcopenia, yet another such disorder whichcan affect the aging individual, is typically characterized by loss ofmuscle mass. End stage wasting disease as described above can develop inindividuals suffering from either cachexia or sarcopenia.

Melanocortin Antagonist Peptides. Antagonist peptides are based onmodifications of the alpha-melanocyte stimulating hormone (α-MSH) coresequence, His-Phe-Arg-Trp (SEQ ID NO:1), and generally include a D-aminoacid at the Phe position, most commonly a D-amino acid with a 1- or2-naphthyl ring or phenyl ring, which may optionally be a substitutedring. Thus U.S. Pat. No. 5,731,408 discloses cyclic lactam heptapeptidesthat are non-specific antagonists for melanocortin receptors MC3-R andMC4-R, and contain either D-Phe(4-I) or D-Nal 2 in place of the Pheresidue. Of particular note is a peptide commonly called SHU9119(Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-NH₂) disclosed in U.S. Pat.No. 5,731,408. SHU9119 has been extensively used in research as areference non-specific melanocortin antagonist. Related cyclic lactamheptapeptides are disclosed in U.S. Pat. No. 6,054,556 which areantagonists for melanocortin receptors MC1-R, MC3-R, MC4-R and MC5-R.These peptides all contain an optionally substituted D-Phe or D-Nal 2 inplace of the Phe residue. All of the peptides disclosed in U.S. Pat.Nos. 5,731,408 and 6,054,556 have a C-terminus NH₂ group, which isconventional for melanocortin-specific peptides.

Other patents teach the use of melanocortin antagonists for treatment ofcachexia and other weight-related disorders. See, for example, U.S. Pat.Nos. 6,716,810; 6,699,873; 6,693,165; 6,613,874; 6,476,187; 6,284,729;6,100,048; and 5,908,609. However, none of these disclose the peptidesof the present invention. U.S. Pat. No. 6,693,165 discloses cyclicheptapeptides and hexapeptides that are asserted to be selective MC4-Rantagonists. These peptides all include a D-amino acid containing anoptionally substituted 1- or 2-naphthyl, 3-benzothienyl or phenyl inplace of the Phe residue in the His-Phe-Arg-Trp (SEQ ID NO:1) coresequence. However, the peptides disclosed in U.S. Pat. No. 6,693,165optionally omit the His in the His-Phe-Arg-Trp (SEQ ID NO:1) sequence,and when the His position is present, it is limited to Lys or His. Eachof the peptides disclosed in U.S. Pat. No. 6,693,165, and the genericformulas given therein, have a C-terminus NH₂ group.

Published U.S. Application 2003/0113263, “Methods and Reagents for UsingMammalian Melanocortin Receptor Antagonists to Treat Cachexia”,discloses a method for characterizing a compound useful for treating ananimal with cachexia, including use of an MC4-R antagonist to treat ananimal with cachexia, and specifically disclosing SHU9119. PublishedU.S. Application 2003/0105024, “Methods and Reagents for Discovering andUsing Mammalian Melanocortin Receptor Agonists and Antagonists toModulate Feeding Behavior in Animals”, discloses SHU9119 as a MCreceptor antagonist used experimentally to stimulate feeding behavior.U.S. Pat. No. 6,476,187, “Methods and Reagents for Discovering and UsingMammalian Melanocortin Receptor Agonists and Antagonists to ModulateFeeding Behavior in Animals”, similarly discloses SHU9119 as a MCreceptor antagonist used experimentally to stimulate feeding behavior.Published U.S. Application 2003/0032791, “Novel Melanocortin-4 ReceptorSequences and Screening Assays to Identify Compounds Useful inRegulating Animal Appetite and Metabolic Rate”, discloses theexperimental use of SHU9119 in various assays. Published U.S.Application 2002/0016291, “Cyclic Peptides as Potent and SelectiveMelanocortin-4 Receptor Antagonists”, discloses SHU9119 as an antagonistat the MC3 and MC4 receptors. In 1977, it was disclosed that SHU9119enhanced feeding behavior. Fan W., Boston B. A., Kesterson R. A., HrubyV. J., Cone R. D. Role of melanocortinergic neurons in feeding and theagouti obesity syndrome. Nature 385:165-168 (1997); see also Rossi M.,Kim M. S., Morgan D. G., et al. A C-terminal fragment of Agouti-relatedprotein increases feeding and antagonizes the effect of alpha-melanocytestimulating hormone in vivo. Endocrinology 139:4428-31 (1998); Wisse B.E., Frayo R. S., Schwartz M. W., Cummings D. E. Reversal of canceranorexia by blockade of central melanocortin receptors in rats.Endocrinology 142:3292-3301 (2001); Marks D. L., Ling N., Cone R. D.Role of the central melanocortin system in cachexia. Cancer Research61:1432-1438 (2001).

There remains a significant need for ligands with high specificity fordiscrete melanocortin receptors, and specifically MC4-R, as well asligands that are antagonists, or optionally inverse agonists, of MC4-R.In order to reduce unintended pharmacological responses, it is desirablethat the ligand be highly specific for the target MC receptor, such asMC4-R. Thus it is desirable that the binding affinity of a ligand forMC4-R be higher, such as at least about ten times higher, for MC4-R thanfor other MC receptors. High affinity peptide ligands of melanocortinreceptors can be used to exploit varied physiological responsesassociated with the melanocortin receptors, either as antagonists orinverse agonists. For example, antagonists or inverse agonists of MC4-Rcan be used to treat eating disorders, wasting diseases and cachexia. Inaddition, melanocortin receptors have an effect on the activity ofvarious cytokines, and high affinity peptide ligands of melanocortinreceptors can be used to regulate cytokine activity. Thus such peptideligands may further be used for treatment of inflammation and otherimmune disorders.

BRIEF SUMMARY OF THE INVENTION

The invention provides a cyclic peptide of the structural formula:

wherein:

-   -   R₁ is H, NH₂,

-   -   R₂ is —C(═O)—NH—, —NH—C(═O)—, —S—, or —S—S—;    -   R₃ is 4-imidazolyl or 3-indolyl;    -   R_(4a) and R_(4b) are each optional ring substituents, and when        one or both are present, are the same or different and        independently hydroxyl, halogen, alkyl, or aryl groups attached        directly or through an ether linkage;    -   R₅ is —NH₂ or —NH(C═NH)NH₂;    -   R₆ is 1- or 2-naphthyl or 3-indolyl, optionally with one or two        ring substituents, and when one or both ring substitutents are        present, are the same or different and independently hydroxyl,        halogen, alkyl, or aryl groups attached directly or through an        ether linkage;

-   -   R₈ is H, NH₂, a lower aliphatic C₁ to C₄ linear or branched        alkyl chain, a C₁ to C₄ aralkyl, or a C₁ to C₄ omega amino        derivative;    -   R₉ is H, a lower aliphatic C₁ to C₄ linear or branched alkyl        chain, a C₁ to C₄ aralkyl, or a C₁ to C₄ omega amino derivative;    -   R₁₀ is an aliphatic L- or D-amino acid, an N-acylated L- or        D-amino acid or a linear or branched C₁ to C₁₇ alkyl, aryl,        heteroaryl, alkene, alkenyl, or aralkyl chain;    -   R₁₁ and R₁₂ are each independently H or a C₁ to C₄ linear or        branched alkyl chain, on the proviso that both R₁₁ and R₁₂ are        not H;    -   x is 1 to 4, and y is 1 to 5, provided that x+y is 2 to 7; and    -   z is 2 to 5.

In one embodiment, the cyclic peptide has the structural formula:

wherein R₃ and R₆ are as defined for the peptide of structural formula(I). Representative cyclic peptides of formula (II) include:

-   -   Ac-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH;    -   Ac-cyclo(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH; or    -   Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH.

In another embodiment, the cyclic peptide has the structural formula:

wherein R₃ and R₆ are as defined for the peptide of structural formula(I). Representative cyclic peptides of formula (III) include:

-   -   Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH;    -   Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH; or    -   Ac-Nle-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH.

In another embodiment, the cyclic peptide has the structural formula:

wherein R₃, R₆, R₁₁ and R₁₂ are as defined for the peptide of structuralformula (I). Representative cyclic peptides of formula (IV) include:

-   -   Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₂—CH₃;    -   Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂; or    -   Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃.

In another embodiment, the cyclic peptide has the structural formula:

wherein R₃, R₅, R₆, R₁₁, R₁₂ and z are as defined for the peptide ofstructural formula (I). Representative cyclic peptides of formula (V)include:

-   -   H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₂—CH₃;    -   H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃;    -   H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂;    -   H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-NH—CH₂—CH₃;    -   H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-NH—CH₃; or    -   H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-N(CH₃)₂.

Another embodiment of the present invention further provides apharmaceutical preparation, comprising a cyclic peptide of any offormulas (I) to (V) or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

Yet another embodiment of the present invention provides a method oftreating cachexia. The method includes administration of apharmaceutically sufficient amount of a pharmaceutical preparation asprovided. Yet another embodiment of the present invention provides amethod of treating inflammation and immune-mediated disorders. Themethod includes administration of a pharmaceutically sufficient amountof a pharmaceutical preparation as provided.

In yet another embodiment, the invention provides a cyclic hexapeptidewith a C-terminus hydroxyl or N-alkyl group, wherein the N-alkyl groupcomprises one or two C₁ to C₄ linear or branched alkyl chains, thehexapeptide containing the core sequence His-D-Nal 2-X—Y or Trp-D-Nal2-X—Y, wherein X is an L-amino acid selected from the group consistingof Arg, Lys, Orn, Harg and Hlys and Y is an L- or D-amino acid selectedfrom the group consisting of Nal 1, Nal 2 and Trp, and wherein anyaromatic ring in the core sequence may optionally include one or tworing substituents, and when one or both ring substitutents are present,are the same or different and independently hydroxyl, halogen, alkyl, oraryl groups attached directly or through an ether linkage. In oneembodiment, the cyclic hexapeptide has an N-terminus Ac or NH₂ group.The cyclic hexapeptide may be cyclized by formation of an amide bondbetween an amino group of a side chain of an amino acid in the 1position or an amino group of the N-terminus group of the amino acid inthe 1 position and a side chain carboxyl group of an amino acid residueat the 6 position. Alternatively, the cyclic hexapeptide may be cyclizedby formation of an amide bond between a side chain carboxyl group of anamino acid residue in the 1 position and an amino group of a side chainof an amino acid at the 6 position. In yet another alternative, thecyclic hexapeptide may be cyclized by formation of a covalent bondcomprising an amide, disulfide, thioether, Schiff base, reduced Schiffbase, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond.In a preferred embodiment of the cyclic hexapeptide, the core sequenceis in the 2 to 5 positions and is His-D-Nal 2-X-Nal 2, and is cyclizedthrough the amino acids in the 1 and 6 positions. In another preferredembodiment of the cyclic hexapeptide the core sequence is in the 2 to 5positions and is Trp-D-Nal 2-X-Nal 2, and is cyclized through the aminoacids in the 1 and 6 positions. In yet another preferred embodiment ofthe cyclic hexapeptide the core sequence is in the 2 to 5 positions andis His-D-Nal 2-X-Trp, and is cyclized through the amino acids in the 1and 6 positions. Positions are determined in the conventional manner, bycounting amino acid residue positions from the N-terminus to theC-terminus.

An object of the present invention is to provide a peptide-basedmelanocortin receptor-specific pharmaceutical, wherein the peptide is ahighly-selective MC4-R antagonist or inverse agonist, for use intreatment of cachexia.

Another object of the present invention is to provide a peptide-basedmelanocortin receptor-specific pharmaceutical for use in treatment ofcachexia wherein the peptide has a C-terminus hydroxyl.

Another object of the invention is to provide a peptide-basedmelanocortin receptor-specific pharmaceutical for use in treatment ofcachexia wherein the peptide has a C-terminus N-alkyl group.

Another object of this invention is to provide peptides which are highlyspecific for melanocortin receptor MC4-R and which are antagonists orinverse agonists.

Another object of the present invention is a peptide-based melanocortinreceptor-specific pharmaceutical for use in treatment of inflammationand other immune related disorders.

Yet another object of the present invention is to provide a melanocortinreceptor-specific pharmaceutical for use in treatment whereinadministration of the treatment is via nasal administration.

According to one embodiment of the present invention, there is provideda C-terminus hydroxyl cyclic peptide that is a highly specific MC4-Rantagonist or inverse agonist suitable for-use as a specificpharmaceutical in treatment of eating disorders and which is efficaciousat low doses.

Another embodiment of the present invention provides a C-terminusN-alkyl cyclic peptide that is a highly specific MC4-R antagonist orinverse agonist suitable for use as a specific pharmaceutical intreatment of eating disorders and which is efficacious at low doses.

Another aspect of the present invention provides a highly specific MC4-Rcyclic peptide antagonist or inverse agonist that is effective over asignificant dose range.

Yet another aspect of the present invention provides highly specificMC4-R cyclic peptide antagonists or inverse agonists for use intreatment of eating disorders which, because of increased efficacy atlow doses, may be administered by delivery systems other than artconventional intravenous, subcutaneous or intramuscular injection,including but not limited to oral delivery systems, nasal deliverysystems and mucous membrane delivery systems.

Other objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is a graph of the cumulative 24 hour food intake in male rats,comparing the effect of IV administration of 1 mg/kg of the compound ofExample 12 against the same volume of vehicle; and

FIG. 2 is a graph of the change in body weight at 24 hours in theanimals of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Certain terms as used throughout the specification and claims aredefined as follows.

The terms “bind,” “binding,” “complex,” and “complexing,” as usedthroughout the specification and claims, are generally intended to coverall types of physical and chemical binding, reactions, complexing,attraction, chelating and the like.

The “peptides” of this invention can be a) naturally-occurring, b)produced by chemical synthesis, c) produced by recombinant DNAtechnology, d) produced by biochemical or enzymatic fragmentation oflarger molecules, e) produced by methods resulting from a combination ofmethods a through d listed above, or f) produced by any other means forproducing peptides.

By employing chemical synthesis, a preferred means of production, it ispossible to introduce various amino acids which do not naturally occuralong the chain, modify the N— or C-terminus, and the like, therebyproviding for improved stability and formulation, resistance to proteasedegradation, and the like.

The term “peptide” as used throughout the specification and claims isintended to include any structure comprised of two or more amino acids,including chemical modifications and derivatives of amino acids. Theamino acids forming all or a part of a peptide may be naturallyoccurring amino acids, stereoisomers and modifications of such aminoacids, non-protein amino acids, post-translationally modified aminoacids, enzymatically modified amino acids, constructs or structuresdesigned to mimic amino acids, and the like, so that the term “peptide”includes pseudopeptides and peptidomimetics, including structures whichhave a non-peptidic backbone. The term “peptide” also includes dimers ormultimers of peptides. A “manufactured” peptide includes a peptideproduced by chemical synthesis, recombinant DNA technology, biochemicalor enzymatic fragmentation of larger molecules, combinations of theforegoing or, in general, made by any other method.

The term “amino acid side chain moiety” used in this invention,including as used in the specification and claims, includes any sidechain of any amino acid, as the term “amino acid” is defined herein.This thus includes the side chain moiety present in naturally occurringamino acids. It further includes side chain moieties in modifiednaturally occurring amino acids, such as glycosylated amino acids. Itfurther includes side chain moieties in stereoisomers and modificationsof naturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, derivatized amino acids, constructs or structures designedto mimic amino acids, and the like. For example, the side chain moietyof any amino acid disclosed herein is included within the definition. A“derivative” of an amino acid side chain moiety is included within thedefinition of an amino acid side chain moiety.

The “derivative” of an amino acid side chain moiety includes anymodification to or variation in any amino acid side chain moieties,including a modification of naturally occurring amino acid side chainmoieties. By way of example, derivatives of amino acid side chainmoieties include straight chain or branched, cyclic or noncyclic,substituted or unsubstituted, saturated or unsaturated, alkyl, aryl oraralkyl moieties.

The “amino acids” used in embodiments of the present invention, and theterm as used in the specification and claims, include the knownnaturally occurring protein amino acids, which are referred to by boththeir common three letter abbreviation and single letter abbreviation.See generally Synthetic Peptides: A User's Guide, G. A. Grant, editor,W. H. Freeman & Co., New York (1992), the teachings of which areincorporated herein by reference, including the text and table set forthat pages 11 through 24. As set forth above, the term “amino acid” alsoincludes stereoisomers and modifications of naturally occurring proteinamino acids, non-protein amino acids, post-translationally modifiedamino acids, enzymatically synthesized amino acids, derivatized aminoacids, constructs or structures designed to mimic amino acids, and thelike. Modified and unusual amino acids are described generally inSynthetic Peptides: A User's Guide, cited above; Hruby V. J., Al-obeidiF., Kazmierski W., Biochem. J. 268:249-262 (1990); and Toniolo C., Int.J. Peptide Protein Res. 35:287-300 (1990); the teachings of all of whichare incorporated herein by reference. In addition, the followingabbreviations have the meanings giving:

-   -   7′-amino-heptanoyl-NH₂—(CH₂)₆CO—    -   Harg—Homo arginine    -   Hlys—Homo lysine    -   Nal 1—3-(1-naphthyl)alanine    -   Nal 2—3-(2-naphthyl)alanine

In the listing of peptides according to the present invention,conventional amino acid residues have their conventional meaning asgiven in Chapter 2400 of the Manual of Patent Examining Procedure,8^(th) Ed. Thus, “Nle” is norleucine, “Asp” is aspartic acid, “His” ishistidine, “D-Phe” is D-phenylalanine, “Arg” is arginine, “Trp” istryptophan, “Lys” is lysine, and so on.

The term “alkene” includes unsaturated hydrocarbons that contain one ormore double carbon-carbon bonds. Examples of such alkene groups includeethylene, propene, and the like.

The term “alkenyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one double bond; examplesthereof include ethenyl, 2-propenyl, and the like.

The “alkyl” groups specified herein include those alkyl radicals of thedesignated length in either a straight or branched configuration.Examples of such alkyl radicals include methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl,isohexyl, and the like.

The term “alkynal” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one triple bond; examplesthereof include ethynyl, propynal, butynyl, and the like.

The term “aryl” includes a monocyclic or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents selected from alkyl, haloalkyl,cycloalkyl, alkoxy, alkythio, halo, nitro, acyl, cyano, amino,monosubstituted amino, disubstituted amino, hydroxy, carboxy, oralkoxy-carbonyl. Examples of an aryl group include phenyl, biphenyl,naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the like.

The term “aralkyl” includes a radical —R^(a)R^(b) where R^(a) is analkylene (a bivalent alkyl) group and R^(b) is an aryl group as definedabove. Examples of aralkyl groups include benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like.

The term “aliphatic” includes compounds with hydrocarbon chains, such asfor example alkanes, alkenes, alkynes, and derivatives thereof.

The term “acyl” includes a group RCO—, where R is an organic group. Anexample is the acetyl group CH₃CO—, referred to herein as “Ac”.

A peptide or aliphatic moiety is “acylated” when an alkyl or substitutedalkyl group as defined above is bonded through one or more carbonyl{—(C═O)—} groups. A peptide is most usually acylated at the N-terminus.

An “omega amino derivative” includes an aliphatic moiety with a terminalamino group. Examples of omega amino derivatives include aminoheptanoyl,such as 7′-amino-heptanoyl, and the amino acid side chain moieties ofornithine and lysine.

The term “heteroaryl” includes mono- and bicyclic aromatic ringscontaining from 1 to 4 heteroatoms selected from nitrogen, oxygen andsulfur. 5- or 6-membered heteroaryl are monocyclic heteroaromatic rings;examples thereof include thiazole, oxazole, thiophene, furan, pyrrole,imidazole, isoxazole, pyrazole, triazole, thiadiazole, tetrazole,oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and the like.Bicyclic heteroaromatic rings include, but are not limited to,benzothiadiazole, indole, benzothiophene, benzofuran, benzimidazole,benzisoxazole, benzothiazole, quinoline, benzotriazole, benzoxazole,isoquinoline, purine, furopyridine and thienopyridine.

An “amide” includes compounds that have a trivalent nitrogen attached toa carbonyl group (—CO.NH₂), such as for example methylamide, ethylamide,propylamide, and the like.

An “imide” includes compounds containing an imido group (—CO.NH.CO—).

An “amine” includes compounds that contain an amino group (—NH₂).

A “nitrile” includes compounds that are carboxylic acid derivatives andcontain a (—CN) group bound to an organic group.

The term “halogen” is intended to include the halogen atoms fluorine,chlorine, bromine and iodine, and groups including one or more halogenatoms, such as —CF₃ and the like.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a cyclic peptide of the present inventionand a pharmaceutically acceptable carrier.

A single amino acid, including stereoisomers and modifications ofnaturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, derivatized amino acids, constructs or structures designedto mimic amino acids, and the like, including all of the foregoing, issometimes referred to herein as a “residue.”

By a melanocortin receptor “agonist” is meant a naturally occurringsubstance or manufactured drug substance or composition that caninteract with a melanocortin receptor and initiate a pharmacologicalresponse characteristic of the melanocortin receptor. By a melanocortinreceptor “antagonist” is meant a naturally occurring substance ormanufactured drug substance or composition that opposes the melanocortinreceptor-associated responses normally induced by a melanocortinreceptor agonist agent. By a melanocortin receptor “inverse agonist” ismeant a drug or a compound that stabilizes the inactive conformation ofthe melanocortin receptor and inhibits basal activity.

“Eating disorders” are those related to underweight, cachexia, anorexiaor bulimia of any cause in humans.

“Cachexia” refers to a state of general ill health and malnutrition. Itis often associated with and induced by malignant cancer, cysticfibrosis or AIDS, and is characterized by loss of appetite, loss of bodymass, especially lean body mass, and muscle wasting.

“Anorexia” refers simply to a loss of appetite, whether brought on bymedical, physiological or psychological factors. Anorexia is oftenclosely associated with, and generally contributes to, cachexia seen inpatients with advanced cancers and other conditions.

Cyclic Peptides of the Invention

One embodiment of the present invention provides cyclic peptides whichinclude the core sequence His-D-Nal 2-Arg-Nal 2, Trp-D-Nal 2-Arg-Nal 2,His-D-Nal 2-Arg-Trp or homologs or analogs of the foregoing, includingpeptides with one or more substituted ring groups in the core sequence.In each of the foregoing, Arg may be substituted with Lys. In anotherembodiment the invention provides cyclic peptides which include the coresequence His-D-Nal 2-Arg-Nal 2, Trp-D-Nal 2-Arg-Nal 2, His-D-Nal2-Arg-Trp, or homologs or analogs of the foregoing, includingsubstitution of Lys for Arg, in which the peptide is deamidated, whichis to say that it does not include an —NH₂ group at the C-terminus. In apreferred embodiment, the deamidated α-MSH cyclic peptides of thisinvention have an —OH group at the C-terminus, and are thus a free acidform of cyclic peptide. In an alternative preferred embodiment, thepeptide has a substituted amide, and specifically an N-alkyl group, atthe C-terminus.

Another aspect of the present invention provides certain cyclic peptideswhich are highly specific for a melanocortin receptor, preferably MC4-R,and alternatively for both MC4-R and MC3-R. Most preferably the cyclicpeptides bind to MC4-R with high affinity, with a Ki value of at least100 nM, preferably of at least 10 nM and most preferably from about 0.01nM to about 2 nM. In some embodiments the cyclic peptides arefunctionally inverse agonists with respect to such receptor orreceptors. However, the peptides of this invention need not be inverseagonists. Such peptides can preferably be employed in the treatment ofeating disorders, and may be characterized in part as inducing weightincrease in mammals, including but not limited to rodents, canines andhumans.

The peptide is a cyclic peptide. A cyclic peptide can be obtained byinducing the formation of a covalent bond between an amino group at theN-terminus of the peptide, if provided, and a carboxyl group at theC-terminus, if provided. A cyclic peptide can also be obtained byforming a covalent bond between a terminal reactive group and a reactiveamino acid side chain moiety, or between two reactive amino acid sidechain moieties. A cyclic peptide can also be obtained by forming adisulfide covalent bond between two sulfhydryl group containing aminoacid side chain moieties or a terminal sulfhydryl group and a sulfhydrylgroup in another amino acid side chain moiety. Peptides withlanthionine, cystathionine, or penthionine covalent bonds can also beformed, such as cyclic bonds formed from cysteine, homocysteine orpenicillamine amino acid residues. These bonds are thioether-bridgedbonds. Galande A. K., Spatola A. F. Lett. Pept. Sci. 8:247 (2002),disclosing methods of making such bonds, is incorporated herein byreference. Thus a cyclic peptide can also be obtained by forming athioether covalent bond between two reactive amino acid side chainmoieties or between a terminal reactive group and a reactive amino acidside chain moiety. One skilled in the art would know that the means bywhich a given peptide is made cyclic is determined by the reactivegroups present in the peptide and the desired characteristics of thepeptide.

The cyclic peptides as disclosed in the several embodiments of thepresent invention are characterized, in part, in that the peptides arepreferably highly selective for MC4-R. For example, with SHU9119 theratio of the Ki values for MC4-R to MC3-R is, under the assay conditionsemployed herein, less than about 1:6, the ratio of MC4-R to MC5-R isless than about 1:3, and the ratio of MC4-R to MC1-R is less than about1:7. Other researchers (e.g., Schioth H. B. et al. Peptides18:1009-1013(1997)), while reporting different values, concur thatSHU9119 is non-selective. It may thus be seen that SHU9119 is not highlyselective for MC4-R. By contrast, certain peptides of this invention aresignificantly more selective. The cyclic peptide of Example 16,Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH, has, under the same assayconditions, a ratio of Ki values for MC4-R to MC3-R of approximately1:110, for MC4-R to MC5-R of approximately 1:187, and for MC4-R to MC1-Rof approximately 1:12,095. The cyclic peptide of Example 19,Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃, has, again under thesame assay conditions, a ratio of Ki values for MC4-R to MC3-R ofapproximately 1:54, for MC4-R to MC5-R of approximately 1:101, and forMC4-R to MC1-R of approximately 1:10,531. It may thus be seen that atall pharmaceutically relevant doses the cyclic peptides of thisinvention are highly selective for MC4-R.

The cyclic peptides as disclosed in the several embodiments of theinvention are further characterized in that they are preferable notagonists for any MC receptor, and are preferably either inactive orantagonists as to all MC receptors other than MC4-R. All cyclic peptidesof the invention are functional antagonists as to MC4-R. Certainpeptides of the invention are partial agonists or agonists, as to MC1-R;these are peptides with a His-D-Nal 2-Arg-Nal 2, His-D-Nal 2-Arg-Trp, orTrp-D-Nal 2-Arg-Nal 2 core sequence and a C-terminus N-alkyl orhydroxyl. However, these peptides are functional antagonists as to MC3-Rand MC4-R.

Peptide Synthesis

The cyclic peptides as disclosed in the several embodiments of thisinvention may be readily synthesized by any known conventional procedurefor the formation of a peptide linkage between amino acids. Suchconventional procedures include, for example, any solution phaseprocedure permitting a condensation between the free alpha amino groupof an amino acid residue having its carboxyl group or other reactivegroups protected and the free primary carboxyl group of another aminoacid residue having its amino group or other reactive groups protected.In a preferred conventional procedure, the cyclic peptides of thisinvention may be synthesized by solid-phase synthesis and purifiedaccording to methods known in the art. Any of a number of well-knownprocedures utilizing a variety of resins and reagents may be used toprepare the cyclic peptides of this invention.

The process for synthesizing the cyclic peptides may be carried out by aprocedure whereby each amino acid in the desired sequence is added oneat a time in succession to another amino acid residue or by a procedurewhereby peptide fragments with the desired amino acid sequence are firstsynthesized conventionally and then condensed to provide the desiredpeptide. The resulting peptide is then cyclized to yield a cyclicpeptide of the invention.

Solid phase peptide synthesis methods are well known and practiced inthe art. In such methods the synthesis of peptides of the invention canbe carried out by sequentially incorporating the desired amino acidresidues one at a time into the growing peptide chain according to thegeneral principles of solid phase methods. These methods are disclosedin numerous references, including Merrifield R. B., Solid phasesynthesis (Nobel lecture). Angew. Chem. 24:799-810 (1985) and Barany etal., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross E. andMeienhofer J., Eds. Academic Press 1-284 (1980).

In chemical syntheses of peptides, reactive side chain groups of thevarious amino acid residues are protected with suitable protectinggroups, which prevent a chemical reaction from occurring at that siteuntil the protecting group is removed. Also common is the protection ofthe alpha amino group of an amino acid residue or fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the alpha amino protecting group to allow a subsequent reaction totake place at that site. Specific protecting groups have been disclosedand are known in solid phase synthesis methods and solution phasesynthesis methods.

Alpha amino groups may be protected by a suitable protecting group,including a urethane-type protecting group, such as benzyloxycarbonyl(Z) and substituted benzyloxycarbonyl, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz);aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, andallyloxycarbonyl. Fmoc is preferred for alpha amino protection.

Guanidino groups may be protected by a suitable protecting group, suchas nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc),adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) andBoc. Pmc is a preferred protecting group for Arg.

The peptides of the invention described herein were prepared using solidphase synthesis, such as by means of a Symphony Multiplex PeptideSynthesizer (Rainin Instrument Company) automated peptide synthesizer,using programming modules as provided by the manufacturer and followingthe protocols set forth in the manufacturer's manual.

Solid phase synthesis is commenced from the C-terminal end of thepeptide by coupling a protected alpha amino acid to a suitable resin.Such starting material is prepared by attaching an alpha amino-protectedamino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang)resin or a 2-chlorotrityl chloride resin, by an amide bond between anFmoc-Linker, such asp-[(R,S)-α-[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyaceticacid (Rink linker) to a benzhydrylamine (BHA) resin, or by other meanswell known in the art. Fmoc-Linker-BHA resin supports are commerciallyavailable and generally used when feasible. The resins are carriedthrough repetitive cycles as necessary to add amino acids sequentially.The alpha amino Fmoc protecting groups are removed under basicconditions. Piperidine, piperazine, diethylamine, or morpholine (20-40%v/v) in N,N-dimethylformamide (DMF) may be used for this purpose.

Following removal of the alpha amino protecting group, the subsequentprotected amino acids are coupled stepwise in the desired order toobtain an intermediate, protected peptide-resin. The activating reagentsused for coupling of the amino acids in the solid phase synthesis of thepeptides are well known in the art. After the peptide is synthesized, ifdesired, the orthogonally protected side chain protecting groups may beremoved using methods well known in the art for further derivatizationof the peptide.

Reactive groups in a peptide can be selectively modified, either duringsolid phase synthesis or after removal from the resin. For example,peptides can be modified to obtain N-terminus modifications, such asacetylation, while on resin, or may be removed from the resin by use ofa cleaving reagent and then modified. Methods for N-terminusmodification, such as acetylation, or C-terminus modification, such asintroduction of an N-acetyl group, are known in the art. Similarly,methods for modifying side chains of amino acids are well known to thoseskilled in the art of peptide synthesis. The choice of modificationsmade to reactive groups present on the peptide will be determined, inpart, by the characteristics that are desired in the peptide.

The peptide can, in one embodiment, be cyclized prior to cleavage fromthe peptide resin. For cyclization through reactive side chain moieties,the desired side chains are deprotected, and the peptide suspended in asuitable solvent and a cyclic coupling agent added. Suitable solventsinclude, for example DMF, dichloromethane (DCM) or1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include,for example, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU),benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP),benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate(PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TATU),2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU) or N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole(DCCI/HOBt). Coupling is conventionally initiated by use of a suitablebase, such as N,N-diispropylethylamine (DIPEA), sym-collidine orN-methylmorpholine (NMM).

Following cleavage of peptides from the solid phase following theirsynthesis, the peptide can be purified by any number of methods, such asreverse phase high performance liquid chromatography (RP-HPLC), using asuitable column, such as a C₁₈ column. Other methods of separation orpurification, such as methods based on the size or charge of thepeptide, can also be employed. Once purified, the peptide can becharacterized by any number of methods, such as high performance liquidchromatograph (HPLC), amino acid analysis, mass spectrometry, and thelike.

Peptides of the present invention with a substituted amide derivativeC-terminus, typically an N-alkyl group, are prepared by solid phasesynthesis commenced from the C-terminal end of the peptide by coupling aprotected alpha amino acid to a suitable resin. Such methods forpreparing substituted amide derivatives on solid phase have beendescribed in the art. See, for example, Barn D. R., Morphy J. R., ReesD. C. Synthesis of an array of amides by aluminum chloride assistedcleavage of resin-bound esters. Tetrahedron Lett. 37, 3213-3216 (1996);DeGrado W. F. Kaiser E. T. Solid-phase synthesis of protected peptideson a polymer bound oxime: Preparation of segments comprising thesequences of a cytotoxic 26-peptide analogue. J. Org. Chem. 47:3258-3261(1982). Such starting material can be prepared by attaching an alphaamino-protected amino acid by an ester linkage to a p-benzyloxybenzylalcohol (Wang) resin by well known means. The peptide chain is grownwith the desired sequence of amino acids, the peptide cyclized and thepeptide-resin treated with a solution of appropriate amine and aluminumchoride (such as methyl amine, dimethyl amine, ethylamine, and so on) indichloromethane. The resulting peptide amide derivative is released insolution from the resin. The resin is filtered and the peptide amidederivative recovered by concentration of solvent followed byprecipitation with ether. The crude peptide is dried and remaining aminoacid side chain protective groups cleaved using trifluoroacetic acid(TFA) in the presence of water and 1,2-ethanedithiol (EDT). The finalproduct is precipitated by adding cold ether and collected byfiltration. Final purification is by RP-HPLC using a C₁₈ column.

Formulation and Utility

The cyclic peptides disclosed herein can be used for both medicalapplications and animal husbandry or veterinary applications. Typically,the product is used in humans, but may also be used in other mammals.The term “patient” is intended to denote a mammalian individual, and isso used throughout the specification and in the claims. The primaryapplications of this invention involve human patients, but thisinvention may be applied to laboratory, farm, zoo, wildlife, pet, sportor other animals.

In general, the cyclic peptides of this invention may be synthesized bysolid-phase synthesis and purified according to methods known in theart. Any of a number of well-known procedures utilizing a variety ofresins and reagents may be used to prepare the cyclic peptides of thisinvention.

Salt Form of Cyclic Peptides. The cyclic peptides of this invention maybe in the form of any pharmaceutically acceptable salt. The term“pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic bases or acids including inorganicor organic bases and inorganic or organic acids. Salts derived frominorganic bases include aluminum, ammonium, calcium, copper, ferric,ferrous, lithium, magnesium, manganic salts, manganous, potassium,sodium, zinc, and the like. Particularly preferred are the ammonium,calcium, lithium, magnesium, potassium, and sodium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

When the cyclic peptide of the present invention is basic, acid additionsalts may be prepared from pharmaceutically acceptable non-toxic acids,including inorganic and organic acids. Such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Acid addition salts of thepeptides of this invention are prepared in a suitable solvent from thepeptide and an excess of an acid, such as hydrochloric, hydrobromic,sulfuric, phosphoric, acetic, trifluoroacetic, citric, tartaric, maleic,succinic or methanesulfonic acid. The acetate salt form is especiallyuseful. Where the peptides of embodiments of this invention include anacidic moiety, suitable pharmaceutically acceptable salts may includealkali metal salts, such as sodium or potassium salts, or alkaline earthmetal salts, such as calcium or magnesium salts.

Pharmaceutical Compositions. Another embodiment of the present inventionprovides a pharmaceutical composition that includes a cyclic peptide ofthis invention and a pharmaceutically acceptable carrier. The carriermay be a liquid formulation, and is preferably a buffered, isotonic,aqueous solution. Pharmaceutically acceptable carriers also includeexcipients, such as diluents, carriers and the like, and additives, suchas stabilizing agents, preservatives, solubilizing agents, buffers andthe like, as hereafter described.

The cyclic peptide compositions of the several embodiments of thepresent invention may be formulated or compounded into pharmaceuticalcompositions that include at least one cyclic peptide of this inventiontogether with one or more pharmaceutically acceptable carriers,including excipients, such as diluents, carriers and the like, andadditives, such as stabilizing agents, preservatives, solubilizingagents, buffers and the like, as may be desired. Formulation excipientsmay include polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,polyethylene glycol, manniton, sodium chloride and sodium citrate. Forinjection or other liquid administration formulations, water containingat least one or more buffering constituents is preferred, andstabilizing agents, preservatives and solubilizing agents may also beemployed. For solid administration formulations, any of a variety ofthickening, filler, bulking and carrier additives may be employed, suchas starches, sugars, fatty acids and the like. For topicaladministration formulations, any of a variety of creams, ointments,gels, lotions and the like may be employed. For most pharmaceuticalformulations, non-active ingredients will constitute the greater part,by weight or volume, of the preparation. For pharmaceuticalformulations, it is also contemplated that any of a variety ofmeasured-release, slow-release or time-release formulations andadditives may be employed, so that the dosage may be formulated so as toeffect delivery of a peptide of this invention over a period of time.

In general, the actual quantity of cyclic peptides administered to apatient will vary between fairly wide ranges depending on the mode ofadministration, the formulation used, and the response desired.

In practical use, the cyclic peptides can be combined as the activeingredient in an admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration, for example, oral, parenteral (including intravenous),urethral, vaginal, nasal, buccal, sublingual, or the like. In preparingthe compositions for oral dosage form, any of the usual pharmaceuticalmedia may be employed, such as, for example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likein the case of oral liquid preparations, such as, for example,suspensions, elixirs and solutions; or carriers such as starches,sugars, microcrystalline cellulose, diluents, granulating agents,lubricants, binders, disintegrating agents and the like in the case oforal solid preparations such as, for example, powders, hard and softcapsules and tablets.

Because of their ease of administration, tablets and capsules representan advantageous oral dosage unit form. If desired, tablets may be coatedby standard aqueous or nonaqueous techniques. The amount of activepeptide in such therapeutically useful compositions is such that aneffective dosage will be obtained. In another advantageous dosage unitform, sublingual constructs may be employed, such as sheets, wafers,tablets or the like. The active peptides can also be administeredintranasally as, for example, by liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch or alginic acid; a lubricant such as magnesium stearate;and a sweetening agent such as sucrose, lactose or saccharin. When adosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier such as a fatty oil.

Various other materials may be utilized as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Cyclic peptides may also be administered parenterally. Solutions orsuspensions of these active peptides can be prepared in water suitablymixed with a surfactant such as hydroxy-propylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof in oils. These preparations may optionally contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that it may be administered by syringe. The form must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, a polyol, for example glycerol,propylene glycol or liquid polyethylene glycol, suitable mixturesthereof, and vegetable oils.

Cyclic peptides as disclosed herein may be therapeutically applied bymeans of nasal administration. By “nasal administration” is meant anyform of intranasal administration of any of the cyclic peptides of thisinvention. The peptides may be in an aqueous solution, such as asolution including saline, citrate or other common excipients orpreservatives. The peptides may also be in a dry or powder formulation.

In an alternative embodiment, cyclic peptides may be administereddirectly into the lung. Intrapulmonary administration may be performedby means of a metered dose inhaler, a device allowingself-administration of a metered bolus of a peptide of this inventionwhen actuated by a patient during inspiration.

According to another embodiment of the present invention, cyclicpeptides of this invention may be formulated with any of a variety ofagents that increase effective nasal absorption of drugs, includingpeptide drugs. These agents should increase nasal absorption withoutunacceptable damage to the mucosal membrane. U.S. Pat. Nos. 5,693,608,5,977,070 and 5,908,825, among others, teach a number of pharmaceuticalcompositions that may be employed, including absorption enhancers, andthe teachings of each of the foregoing, and all references and patentscited therein, are incorporated by reference.

If in an aqueous solution, certain cyclic peptides of the presentinvention may be appropriately buffered by means of saline, acetate,phosphate, citrate, acetate or other buffering agents, which may be atany physiologically acceptable pH, generally from about pH 4 to about pH7. A combination of buffering agents may also be employed, such asphosphate buffered saline, a saline and acetate buffer, and the like. Inthe case of saline, a 0.9% saline solution may be employed. In the caseof acetate, phosphate, citrate, acetate and the like, a 50 mM solutionmay be employed. In addition to buffering agents, a suitablepreservative may be employed, to prevent or limit bacteria and othermicrobial growth. One such preservative that may be employed is 0.05%benzalkonium chloride.

It is also possible and contemplated that the cyclic peptide may be in adried and particulate form. In a preferred embodiment, the particles arebetween about 0.5 and 6.0 μm, such that the particles have sufficientmass to settle on the lung surface, and not be exhaled, but are smallenough that they are not deposited on surfaces of the air passages priorto reaching the lung. Any of a variety of different techniques may beused to make dry powder microparticles, including but not limited tomicro-milling, spray drying and a quick freeze aerosol followed bylyophilization. With micro-particles, the peptides may be deposited tothe deep lung, thereby providing quick and efficient absorption into thebloodstream. Further, with such approach penetration enhancers are notrequired, as is sometimes the case in transdermal, nasal or oral mucosaldelivery routes. Any of a variety of inhalers can be employed, includingpropellant-based aerosols, nebulizers, single dose dry powder inhalersand multidose dry powder inhalers. Common devices in current use includemetered dose inhalers, which are used to deliver medications for thetreatment of asthma, chronic obstructive pulmonary disease and the like.Preferred devices include dry powder inhalers, designed to form a cloudor aerosol of fine powder with a particle size that is always less thanabout 6.0 μm.

Microparticle size, including mean size distribution, may be controlledby means of the method of making. For micro-milling, the size of themilling head, speed of the rotor, time of processing and the likecontrol the microparticle size. For spray drying, the nozzle size, flowrate, dryer heat and the like control the microparticle size. For makingby means of quick freeze aerosol followed by lyophilization, the nozzlesize, flow rate, concentration of aerosoled solution and the likecontrol the microparticle size. These parameters and others may beemployed to control the microparticle size.

The cyclic peptides of this invention may be therapeuticallyadministered by means of an injection, typically a deep intramuscularinjection, such as in the gluteal or deltoid muscle, of a time releaseinjectable formulation. In one embodiment, a cyclic peptide of thisinvention is formulated with a polyethylene glycol, such as polyethyleneglycol 3350, and optionally one or more additional excipients andpreservatives, including but not limited to excipients such as salts,polysorbate 80, sodium hydroxide or hydrochloric acid to adjust pH, andthe like. In another embodiment a cyclic peptide of this invention isformulated with a poly(ortho ester), which may be an auto-catalyzedpoly(ortho ester) with any of a variable percentage of lactic acid inthe polymeric backbone, and optionally one or more additionalexcipients. In one embodiment poly(D,L-lactide-co-glycolide)polymer(PLGA polymer) is employed, preferably a PLGA polymer with a hydrophilicend group, such as PLGA RG502H from Boehringer Ingelheim, Inc.(Ingelheim, Germany). Such formulations may be made, for example, bycombining a cyclic peptide of this invention in a suitable solvent, suchas methanol, with a solution of PLGA in methylene chloride, and addingthereto a continuous phase solution of polyvinyl alcohol under suitablemixing conditions in a reactor. In general, any of a number ofinjectable and biodegradable polymers, which are preferably alsoadhesive polymers, may be employed in a time release injectableformulation. The teachings of U.S. Pat. Nos. 4,938,763, 6,432,438, and6,673,767, and the biodegradable polymers and methods of formulationdisclosed therein, are incorporated here by reference. The formulationmay be such that an injection is required on a weekly, monthly or otherperiodic basis, depending on the concentration and amount of cyclicpeptide, the biodegradation rate of the polymer, and other factors knownto those of skill in the art.

Routes of Administration. If it is administered by injection, theinjection may be intravenous, subcutaneous, intramuscular,intraperitoneal or other means known in the art. The peptides of thisinvention may be formulated by any means known in the art, including butnot limited to formulation as tablets, capsules, caplets, suspensions,powders, lyophilized preparations, suppositories, ocular drops, skinpatches, oral soluble formulations, sprays, aerosols and the like, andmay be mixed and formulated with buffers, binders, excipients,stabilizers, anti-oxidants and other agents known in the art. Ingeneral, any route of administration by which the peptides of inventionare introduced across an epidermal layer of cells may be employed.Administration means may thus include administration through mucousmembranes, buccal administration, oral administration, dermaladministration, inhalation administration, nasal administration,urethral administration, vaginal administration, and the like.

Therapeutically Effective Amount. In general, the actual quantity ofcyclic peptide of this invention administered to a patient will varybetween fairly wide ranges depending upon the mode of administration,the formulation used, and the response desired. The dosage for treatmentis administration, by any of the foregoing means or any other meansknown in the art, of an amount sufficient to bring about the desiredtherapeutic effect. Thus a therapeutically effective amount includes anamount of a peptide or pharmaceutical composition of this invention thatis sufficient to therapeutically alleviate feeding disorder in apatient, or to prevent or delay onset or recurrence of the feedingdisorder, or for the management of the feeding disorder in patients withdiseases or syndromes associated with cachexia, including secondary toimmune disorders and cancer.

In general, the cyclic peptides of this invention are highly active. Forexample, the cyclic peptide can be administered at about 0.01, 0.05,0.1, 0.5, 1, 5, 10, 50, 100, or 500 μg/kg body weight, depending on thespecific peptide selected, the desired therapeutic response, the routeof administration, the formulation and other factors known to those ofskill in the art.

Inflammation and Immune-Mediated Disorders. The peptides of thisinvention may further be employed in the treatment of inflammation andimmune-mediated disorders. See, for example, Catania A. et al., TrendsEndocrinol. Metab. 11:304-308 (2000); Gantz I. and Fong T. M., Am. J.Physiol. Endocrinol. Metab. 284:E468-E474 (2003); and Catania A., GattiS., Colombo G., Lipton J. M., Pharmacol. Rev. 56:1-29 (2004); eachincorporated here by reference.

Combination Therapy

It is also possible and contemplated that cyclic peptides according toseveral embodiments of the present invention are used in combinationwith other drugs or agents, particularly in the treatment of cachexia.These other drugs and agents may include agents that induce weight gain,including corticosteroids and progestational agents. In a preferredembodiment of the invention, cyclic peptides of the invention are usedin combination with a therapeutically effective amount of a secondweight gain pharmaceutical agent.

According to another embodiment of the present invention, a method fortreating cachexia is provided. The method includes administering to thepatient having or at risk of having cachexia a therapeutically effectiveamount of a cyclic peptide as disclosed herein in combination with atherapeutically effective amount of another compound that is useful inthe treatment of cachexia.

Thus one object of the present invention is to provide pharmaceuticalcompositions that include 1) a cyclic peptide of one embodiment of thepresent invention and 2) a second compound useful for the treatment ofcachexia.

In an embodiment, the second compound useful for the treatment ofcachexia are preferably selected from but not limited to the groupconsisting of ADP-ribose-polymerase inhibitors, ADP-ribose-transferaseinhibitors, NADase inhibitors, nicotinamide benzamide, theophylline,thymine and analogs thereof; omega-3 fatty acids such as alpha-linolenicacid, stearidonic acid, eicosapentaenoic acid (EPA), docosapentaenoicacid, docosahexaenoic acid or mixtures thereof; branched-chain aminoacids valine, leucine, isoleucine or mixtures thereof, with or withoutreduced levels of tryptophan and 5-hydroxytryptophan; antioxidantsselected from the group comprising beta-carotene, vitamin C, vitamin E,selenium, or mixtures thereof; L-glutamine, vitamin A, vitamin C,vitamin E, and selenium; Azaftig; quinine derivatives including3,5,6-trimethyl-2-(3-pyridyl)methyl-1,4-benzoquinone hydrochloride;interleukin 2; benzaldehyde; 4,6-O-benzylidene-D-glucose;friedelan-3-one; hydrazine sulfate; medroxyprogesterone acetate; beta2-adrenoceptor agonists; corticosteroids such as dexamethasone; Vitor™;Pro-Stat™; megestrol acetate (Megace™); dronabinol (Marinol™); magestrolacetate (Megace™); thalidomide (Thalidomid™); fluoxymesterone(Halotestin™); pentoxifylline (Trental™); cyproheptadine (Periactin™);metoclopramide (Reglan™); total parenteral nutrition; or other MC4-Rantagonists. In another embodiment, the second compound useful for thetreatment of cachexia is somatropin (Serostim™), an injectable form ofhuman growth hormone.

Another embodiment of the present invention provides kits for thetreatment of cachexia. The kits include a first pharmaceuticalcomposition including a cyclic peptide according to one embodiment ofthe present invention, a second pharmaceutical composition comprising asecond compound useful for the treatment of cachexia, and a containerfor the first and second compositions.

INDUSTRIAL APPLICABILITY

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Competitive Inhibition Assay Using [I¹²⁵]-NDP-α-MSH

A competitive inhibition binding assay is conducted using membranesprepared using HEK-293 cells transfected with hMC3-R, hMC4-R or hMC5-Rgene constructs, and B-16 mouse melanoma cells (containing MC1-R), usingrespectively 0.4 nM, 0.2 nM, 0.4 nM or 0.1 nM [I¹²⁵]-NDP-α-MSH (NewEngland Nuclear) in 50 mM HEPES buffer containing 1 mM MgCl₂, 2 mMCaCl₂, and 5 mM KCl, at pH 7.2. The assay tube also contains a chosenconcentration of the test peptide of this invention, typically a 1 μMconcentration, for determining its efficacy in inhibiting the binding of[I¹²⁵]-NDP-α-MSH to its receptor. Non-specific binding is measured bycomplete inhibition of binding of [I¹²⁵]-NDP-α-MSH in the assay with thepresence of 1 μM NDP-α-MSH.

The assay mixture is incubated for 90 minutes at room temperature, thenfiltered and the membranes washed three times with ice cold buffer. Thefilter is dried and counted in a gamma counter for remainingradioactivity bound to the membranes. 100% specific binding is definedas the difference in radioactivity (cpm) bound to cell membranes in theabsence and presence of 1 μM NDP-α-MSH. The cpm obtained in presence oftest peptides is normalized with respect to 100% specific binding todetermine the percent inhibition of [I¹²⁵]-NDP-α-MSH binding. Each assayis conducted in triplicate. The Ki (nM) of certain peptides of theinvention are determined using similar assay protocols and testingpeptides over a wider dose range.

EXAMPLE 2 General Method for EC₅₀ Determination in Functional ActivityAssay

Functional evaluation of peptides at melanocortin receptors is performedby measuring the accumulation of intracellular cAMP in HEK-293 cellsexpressing hMC3-R, hMC4-R or hMC5-R, and in B-16 mouse melanoma cellsexpressing MC1-R. Cells suspended in Earle's Balanced Salt Solutioncontaining 10 mM HEPES (pH 7.5), 5 mM MgCl₂, 1 mM glutamine, 0.1%albumin and 0.6 mM 3-isobutyl-1-methyl-xanthine, a phosphodiesteraseinhibitor, are plated in 96 well plates at a density of 0.5×10⁵ cellsper well. Cells are incubated with the test peptides in the presence orabsence of α-MSH for 1 hour at 37° C. cAMP levels in the cell lysatesare measured using the EIA kit (Amersham). Data analysis and EC₅₀ valuesare determined using nonlinear regression analysis with Prism Graph-Padsoftware.

EXAMPLE 3 Functional Status

The agonist/antagonist status with respect to MC1-R, MC4-R and MC5-R ofcertain peptides of the invention is determined. Antagonistic activityis determined by measuring the inhibition of α-MSH-induced orNDP-α-MSH-induced cAMP levels following exposure to the peptides as inthe preceding descriptions.

Assay for agonist. Evaluation of the molecules to elicit a functionalresponse in HEK-293 cells expressing hMC4-R for agonistic activity isdone by measuring the accumulation of intracellular cAMP followingtreatment. Confluent HEK-293 cells over-expressing MC4-R are detached byenzyme free cell suspension buffer. Cells are suspended in Earle'sBalanced Salt Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl₂, 1 mMglutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine, aphosphodiesterase inhibitor. The cells are plated in a 96 well plates ata density of 0.5×10⁵ cells per well and pre-incubated for 30 minutes.The cells are then challenged with the test peptides dissolved indimethylsulfoxide (DMSO) at a concentration range of 0.05-5000 nM in atotal assay volume of 200 μL for 1 hour at 37° C. The concentration ofDMSO is always held at 1% in the assay mixture. NDP-α-MSH is used as thereference agonist. At the end of the incubation period the cells aredisrupted by the addition of 50 μL lysis buffer from the cAMP EIA kit(Amersham). Complete rupture of the cells is ensured by pipetting thecells up and down multiple times. cAMP levels in the cell lysates aremeasured after appropriate dilution using the EIA kit (Amersham) method.Data analysis and EC₅₀ values are determined by using nonlinearregression analysis with the Prism Graph-Pad software. Peptides at aconcentration of 5000 nM with a response ratio compared to NDP-α-MSH of0.7 and above are classified as full agonists. Peptides with a ratiofrom 0.1 to 0.7 are classified as partial agonists. Peptides with aresponse ratio of less than 0.1 are evaluated for antagonistic activity.

Assay for neutral antagonist. Peptides with a high affinity for bindingto MC4-R membranes but with less efficiency (EC₅₀>1000 nM) and lowresponse ratio (<0.1) are analyzed for their ability to antagonize thestimulatory effect of the agonist NDP-α-MSH. These studies are carriedout in HEK-293 cells expressing hMC4-R. Cells are incubated with thepeptides in the presence of the agonist NDP-α-MSH and the extent ofantagonism is measured by the decrease in intracellular cAMPconcentrations. Screening the peptides for antagonists is done at asingle concentration of NDP-α-MSH (1.0 nM) over a peptide concentrationrange of 0.5-5000 nM. Studies are extended further in cases of peptidesexhibiting strong antagonism to derive the pA₂ value from Schild'sanalysis.

Experimental details are similar to the analysis for agonistic activityand are described above. Briefly, cells are pre-incubated for 30 minuteswith the test peptides at concentrations between 0.5 nM and 5000 nM. Thecells are then stimulated with NDP-α-MSH at a concentration of 1 nM for1 hour. For Schild's analysis, the interactions are studied using atleast 3 concentrations of the peptides, separated by a log unit, over afull range of the agonist (0.005-5000 nM). cAMP levels is measured inthe cell lysates after appropriate dilution. Nonlinear regressionanalysis with the Prism Graph-Pad software is used for Schild's analysisand to obtain EC₅₀ values. pA₂ values are derived from the Schild'splot.

Assay for inverse agonist. Peptides that have a weak EC₅₀ value(EC₅₀>1000 nM) or a low response ratio (<0.1) are also investigated fortheir ability to act as inverse agonists, i.e. to decrease the basal orconstitutive level of cAMP in HEK-293 cells expressing hMC4-R receptors.The experimental protocol is essentially the same a described above. Thecells are exposed to the test peptides over a concentration range of0.05 nM to 5000 nM for 1 hour at 37° C. Agouti-related protein (AgRP) ora biologically active fragment of Agouti protein, such as AgRP (83-132)(Ser-Ser-Arg-Arg-Cys-Val-Arg-Leu-His-Glu-Ser-Cys-Leu-Gly-Gln-Gln-Val-Pro-Cys-Cys-Asp-Pro-Cys-Ala-Thr-Cys-Tyr-Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys-Tyr-Cys-Arg-Lys-Leu-Gly-Thr-Ala-Met-Asn-Pro-Cys-Ser-Arg-Thr(SEQ ID NO:2)) is used as the reference inverse agonist. Data analysisand EC₅₀ values are determined by using nonlinear regression analysiswith the Prism Graph-Pad software.

EXAMPLE 4 ICV Food Intake and Body Weight Change

Change in food intake and body weight is evaluated for selectedpeptides. Rats with indwelling intracerebroventricular cannulas (ICVrats) are obtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.).Animals are individually housed in conventional plexiglass hanging cagesand maintained on a controlled 12 hour on/off light cycle. Water andpowdered (LabDiet, 5P00 Prolab RMH 3000) or pelleted (Harlan Teklad 201818% Protein Rodent Diet) food is provided ad libitum. For 1 week beforetreatment, 24-hour food intake and body weight change is recorded toassess a baseline for the group during vehicle treatment. The rats aredosed ICV with vehicle or selected cyclic peptides (0.3-3 nmol). Thechanges in body weight and food intake for the 24 hour period afterdosing are determined. The changes in body weight and food intake forthe 48 hour and 72 hour periods after dosing are also measured todetermine reversal of changes in body weight and food intake effectsback to baseline levels.

EXAMPLE 5 IV and IP Food Intake and Body Weight Change

Change in food intake and body weight is evaluated for selectedpeptides. Male Sprague-Dawley rats are obtained from Taconic(Germantown, N.Y.). Animals are individually housed in conventionalplexiglass hanging cages and maintained on a controlled 12 hour on/offlight cycle. Water and powdered (LabDiet, 5P00 Prolab RMH 3000) orpelleted (Harlan Teklad 2018 18% Protein Rodent Diet) food is providedad libitum. For 1 week before treatment, 24-hour food intake and bodyweight change is recorded to assess a baseline for the group duringvehicle treatment. The rats are dosed IV or IP with vehicle or selectedpeptides (0.5-3 mg/kg). The changes in body weight and food intake forthe 24 hour period after dosing are determined. The changes in bodyweight and food intake for the 48 hour and 72 hour periods after dosingare also measured to determined reversal of changes in body weight andfood intake effects back to baseline levels.

EXAMPLE 6 Behavioral Satiety Sequence

Male Sprague-Dawley rats are maintained on a restricted diet of 20 gpowdered food per day. Food is presented at the same time during thelights-on period, dosed with either saline or the test peptide 2 hoursbefore presentation of food and the start of observation. Pre-weighedbowls containing 20 g of food are presented and the behavior of the ratsis observed for 1 hour. Behavioral observations are divided into 3categories: Feeding, Active (includes grooming, drinking andsniffing/exploration), and Resting (decreased activity and sleep). Theamount of time spent in each behavior is recorded. The amount of foodintake is determined after the observation period.

EXAMPLE 7 Conditioned Taste Avoidance

Male Sprague-Dawley rats are adapted to a restricted drinking period of30 minutes per day during lights on and are provided with pelleted chowad libitum. In laboratory animals the administration of LiCl conditionsan aversion to the novel and favorable taste of saccharin (Seeley R. J.,Blake K., Rushing P. A. et al. The role of CNS glucagons-like peptide-1(7-36) amide receptors in mediating the visceral illness effects oflithium chloride. J. Neurosci. 20:1616-1621 (2000)). To conditionanimals, an injection of LiCl or test peptide is administeredimmediately after the initial presentation of a 0.1% solution ofsaccharin. Two days later, saccharin solution is again presented andfluid intake determined. A decrease in drinking the saccharin solutionsuggests development of a conditioned taste aversion.

EXAMPLE 8 Lipopolysaccharide-Induced Cachexia Model

Rats with indwelling intracerebroventricular cannulas (ICV rats) areobtained from Hilltop Lab Animals, Inc. (Scottdale, Pa.). Animals areindividually housed in conventional plexiglass hanging cages andmaintained on a controlled 12 hour on/off light cycle. Water andpowdered (LabDiet, 5P00 Prolab RMH 3000) or pelleted (Harlan Teklad 201818% Protein Rodent Diet) food are provided ad libitum.Lipopolysaccharide (LPS) (E. Coli 055:B5, Sigma Chemical Co.) isdissolved in normal saline and administered i.p. For the first LPSinjection, male animals aged 6-7 weeks are used. In an identical repeatexperiment, female animals, aged 5 weeks are used. Animals have basalfeeding monitored for two days, and then during each twelve hour periodfollowing an i.p. saline injection prior to injection of 100 μg/kg ofLPS. Certain peptides of the invention are administered, and 50 μg/kgLPS are administered 3 hours later. A second dose of 100 μg/Kg LPS isgiven 60 hours after the first dose in the second experiment. No food isavailable between peptide administration and LPS administration.Starting after LPS administration, feeding is measured every 6 hours for24 hours, then every 12 hours for 48 more hours.

In the sham group, basal feeding is measured every six hours in two ageand sex-matched groups after simulated ICV injection and i.p. salineinjection. Twenty-four hours later, selected peptides are administered,and LPS is administered i.p. 3 hours later. Feeding is measured every 6hours for 24 hours, then every 12 hours for 48 more hours. Thedifference between feeding curves in the two groups is expressed both asweight normalized intake and as a percent of basal feeding vs.post-saline and sham ICV injection.

EXAMPLE 9 Tumor-induced Cachexia Model

Lewis lung carcinoma (LLC) cells and Englebreth-Holm-Swarm Sarcoma (EHS)tumors are maintained either as a primary culture in DMEM with 10% fetalbovine serum or in vivo, respectively, as recommended by the supplier.LLC tumor cells are harvested during exponential growth of the culture,washed in Hanks balanced salt solution, and cells are injectedsubcutaneously into the upper flank of the animals. EHS sarcoma tissueis dissected from a donor animal, and an approximately 3 mm cube oftissue is implanted subcutaneously above the rear flank. Sham operatedanimals receive an implant of a similar amount of donor muscle tissue.In all cases, the time of appearance of a tumor mass is noted, and allanimals are found to have a palpable tumor within four (LLC) or eight(EHS) days of the start of the experiment. At the time of sacrifice,tumors are dissected away from surrounding tissue and weighed. Grossexamination of all organs does not reveal the presence of any observablemetastasis. Trunk blood is collected at the time of sacrifice formeasurement of serum leptin with a rat leptin radioimmunoassay kit.

Animals are individually housed in conventional plexiglass hanging cagesand maintained on a controlled 12 hour on/off light cycle. The effectsof administration of certain peptides of the invention in animals withhypophagia and weight loss due to the presence of a growing sarcoma areexamined. In an initial experiment, daily food intake and weight arefollowed until the tumor-bearing animals have food intake that is 75-80%of basal for three consecutive days. On average this occurs on day 12post-implant, or four days after a palpable tumor is present. ICVinjection of the selected peptides is performed and animals aremonitored to assess the change in food intake.

In a second experiment the ability of selected peptides to prevent theonset of cachexia and maintain normal feeding and growth is tested.Animals are examined daily for the presence of a palpable tumor, withall animals having tumors by day 14 post implantation, and none prior today 12. Animals are then injected with selected peptides or a sham every48 hours until sacrifice. A sham-tumor implanted group is included forcomparison and is also given the peptides.

Differences between feeding, activity, and water consumption curves inall experiments are analyzed by two-way, repeated measure ANOVA withtime and treatment as the measured variables. Final tumor and bodyweights are analyzed by Student's t-test when two groups are included,or one way ANOVA with post-hoc analysis when three groups are included.Data sets are analyzed for statistical significance using either thePRISM software package (GraphPad) for ANOVA with repeated measures, orin EXCEL (Microsoft) using Student's t-test.

EXAMPLE 10 Determination of Mass and Nuclear Magnetic Resonance Analysis

The mass values of peptides of the invention are determined using aWaters MicroMass ZQ device utilizing a positive mode. Massdeterminations are compared with calculated values and expressed in theform of mass weight plus one (M+1 or M+H).

Proton NMR data is obtained using a Bruker 300 MHz spectrometer. Thespectra are obtained after dissolving peptides in a deuteriated solventsuch as chloroform, DMSO, or methanol as appropriate.

EXAMPLE 11 Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH

The peptide Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH wassynthesized by conventional peptide synthesis methods. The formulaweight was determined to be 1189. Competitive inhibition testing and Ki(nM) of the peptide was measured following the method of Example 1.Functional status of the peptide was determined following the methods ofExamples 2 and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 3 10 0.6 3

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was a partial agonist as to MC1-R with anEC₅₀ (nm) of 57, and was an antagonist as to MC3-R and MC4-R. In testsfor functional antagonism as in Example 3, a pA₂ (M) value as to MC4-Rof 7.95 was determined.

EXAMPLE 12 Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH

The peptide Ac-Nle-cyclo(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH wassynthesized by conventional peptide synthesis methods. The formulaweight was determined to be 1200. Competitive inhibition testing and Ki(nM) of the peptide was measured following the method of Example 1.Functional status of the peptide was determined following the methods ofExamples 2 and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 0.2 0.3 0.02 0.2

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was a partial agonist as to MC1-R with anEC₅₀ (nm) of 5, and was an antagonist as to MC3-R and MC4-R. In testsfor functional antagonism as in Example 3, a pA₂ (M) value as to MC4-Rof 8.12 was determined.

FIG. 1 illustrates the cumulative increase in food intake, in grams, inrats administered the compound of Example 12 compared to vehicle alone.Rats were administered 1 mg/kg of the peptide of Example 12 as inExample 5, and food intake was measured at selected times for a 24 hourperiod. Briefly, male Sprague-Dawley rats (300-350 g) were individuallyhoused in shoe box cages with a 12 hr light/dark period. Food intake andbody weights were monitored for 24 hours prior to the start of thestudy. Rats were randomized by body weight and then dosed IV just beforelights-off with the compound of Example 12 or the same volume ofvehicle. A pre-weighed amount of food was provided and food intake wasdetermined at 2, 4, 20 and 24 hours. In FIG. 1, “*” indicates aprobability of p<0.05, and “**” indicates a probability of p<0.01. FIG.2 shows the cumulative change in body weight for the animals of FIG. 1at 24 hours.

EXAMPLE 13 Ac-Nle-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH

The peptide Ac-Nle-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH wassynthesized by conventional peptide synthesis methods. The formulaweight was determined to be 1249. Competitive inhibition testing and Ki(nM) of the peptide was measured following the method of Example 1.Functional status of the peptide was determined following the methods ofExamples 2 and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 119 4 0.2 0.9

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was a partial agonist as to MC1-R and anantagonist as to MC3-R and MC4-R. In tests for functional antagonism asin Example 3, a pA₂ (M) value as to MC4-R of 7.59 was determined.

EXAMPLE 14 Ac-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH

The peptide Ac-cyclo(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH was synthesized byconventional peptide synthesis methods. The formula weight wasdetermined to be 1076. Competitive inhibition testing and Ki (nM) of thepeptide was measured following the method of Example 1. Functionalstatus of the peptide was determined following the methods of Examples 2and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 181 55 2 191

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was an agonist as to MC1-R with an EC₅₀ (nm)of 4420, and was an antagonist as to MC3-R and MC4-R, with an EC₅₀ (nm)as to MC3-R of 1.0. In tests for functional antagonism as in Example 3,a pA₂ (M) value as to MC4-R of 6.73 was determined.

EXAMPLE 15 Ac-cyclo(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH

The peptide Ac-cyclo(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH was synthesizedby conventional peptide synthesis methods. The formula weight wasdetermined to be 1088. Competitive inhibition testing and Ki (nM) of thepeptide was measured following the method of Example 1. Functionalstatus of the peptide was determined following the methods of Examples 2and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 143 6 2 89

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was a partial agonist as to MC1-R with anEC₅₀ (nm) of 2153, and was an antagonist as to MC3-R and MC4-R. In testsfor functional antagonism as in Example 3, a pA₂ (M) value as to MC4-Rof 8.0 was determined.

EXAMPLE 16 Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH

The peptide Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH was synthesizedby conventional peptide synthesis methods. The formula weight wasdetermined to be 1137. Competitive inhibition testing and Ki (nM) of thepeptide was measured following the method of Example 1. Functionalstatus of the peptide was determined following the methods of Examples 2and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 1098 10 0.1 17

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was a partial agonist as to MC1-R with anEC₅₀ (nm) of >1000 and an antagonist as to MC3-R and MC4-R. In tests forfunctional antagonism as in Example 3, a pA₂ (M) value as to MC4-R of8.45 was determined.

EXAMPLE 17 Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₂—CH₃

The peptide Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₂—CH₃ wassynthesized by conventional peptide synthesis methods. The formulaweight was determined to be 1163. Competitive inhibition testing and Ki(nM) of the peptide was measured following the method of Example 1.Functional status of the peptide was determined following the methods ofExamples 2 and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 194 1 0.03 3

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was a partial agonist as to MC1-R with anEC₅₀ (nm) of >1000 and an antagonist as to MC3-R and MC4-R. In tests forfunctional antagonism as in Example 3, a pA₂ (M) value as to MC4-R of8.76 and pA₂ (M) value as to MC3-R of 8.04 was determined.

EXAMPLE 18 Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂

The peptide Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂ wassynthesized by conventional peptide synthesis methods. The formulaweight was determined to be 1163. Competitive inhibition testing and Ki(nM) of the peptide was measured following the method of Example 1.Functional status of the peptide was determined following the methods ofExamples 2 and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 107 1 0.03 4

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide was inactive as to MC1-R and an antagonistas to MC3-R and MC4-R. In tests for functional antagonism as in Example3, a pA₂ (M) value as to MC4-R of 8.69 and pA₂ (M) value as to MC3-R of7.29 was determined.

EXAMPLE 19 Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃

The peptide Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃ wassynthesized by conventional peptide synthesis methods. The formulaweight was determined to be 1149. Competitive inhibition testing and Ki(nM) of the peptide was measured following the method of Example 1.Functional status of the peptide was determined following the methods ofExamples 2 and 3.

Ki (nM) MC1-R MC3-R MC4-R MC5-R 643 3 0.06 6

In a cAMP assay for determination of agonist/antagonist status, it wasdetermined that the peptide at 1 μM concentration was inactive at MC1-R,and an antagonist as to MC3-R and MC4-R. In tests for functionalantagonism as in Example 3, a pA₂ (M) value as to MC4-R of 8.9 and pA₂(M) value as to MC3-R of 8.07 was determined.

EXAMPLE 20-25 Additional Peptides

The following peptides were synthesized by conventional peptidesynthesis methods:

-   -   20. H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₂—CH₃    -   21. H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃    -   22. H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂    -   23. H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-NH—CH₂—CH₃    -   24. H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-NH—CH₃    -   25. H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-N(CH₃)₂

Competitive inhibition and Ki (nM) of the peptides of Examples 20-25 aremeasured following the method of Example 1. Functional status of thepeptides of Examples 20-25 are determined following the methods ofExamples 2 and 3.

EXAMPLE 26 ICV Feeding Studies

A series of ICV feeding studies were conducted on rats. All animals weredosed on the first day with saline ICV, and given a pre-weighed foodbowl with food weight recorded at 2 and 21 hours post-ICV injection. Onday 2, animals were randomized based on the 21 hour food consumption,with animals eliminated due to low food consumption or food spills.Animals were dosed with vehicle (saline), a positive control (SHU9119 at1 nmol) or peptides of the invention (at 0.3 or 3 nmol). Food weightswere again recorded at 2, 4, 21 and 24 hours post-icv injection. In mostinstances, multiple different tests were conducted which each groupcontaining between 8 and 12 members; the lowest value is shown below.Values are shown as percent increase (decrease) in aggregate foodintake. “ND” means no test was conducted at that dose level.

Percent Increase (Decrease) In Aggregate Food Intake After ICVAdministration of Peptide Compared to Vehicle (Saline) Peptide of: 0.3nmol 1 nmol Example 11 ND 24% Example 12 36% 48% Example 13 ND (4)%Example 14 ND 16% Example 15 ND 32% Example 16  0% 4% Example 17 32% 20%Example 18 24% 24% Example 19 28% 32%

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents. The entire disclosures of allreferences, applications, patents, and publications cited above arehereby incorporated by reference.

1. A synthetic cyclic peptide of the structural formula:

wherein: R₁ is H, NH₂, or

R₂ is —C(═O)—NH— or —NH—C(═O)—; R₃ is 4-imidazolyl or 3-indolyl; R_(4a)and R_(4b) are each optional ring substituents, and when one or both arepresent, are the same or different and independently hydroxyl, halogen,alkyl, or aryl groups attached directly or through an ether linkage; R₅is —NH₂ or —NH(C═NH)NH₂; R₆ is 1- or 2-naphthyl or 3-indolyl, optionallywith one or two ring substituents, and when one or both ringsubstituents are present, are the same or different and independentlyhydroxyl, halogen, alkyl, or aryl groups attached directly or through anether linkage;

R₈ is H, NH₂, a lower aliphatic C₁ to C₄ linear or branched alkyl chain,a C₁ to C₄ aralkyl, or a C₁ to C₄ aliphatic moiety with a terminal aminogroup; R₉ is H, a lower aliphatic C₁ to C₄ linear or branched alkylchain, a C₁ to C₄ aralkyl, or a C₁ to C₄ aliphatic moiety with aterminal amino group; R₁₀ is an aliphatic L- or D-amino acid, anN-acylated L- or D-amino acid or a linear or branched C₁ to C₁₇ alkyl,aryl, heteroaryl, alkene, alkenyl, or aralkyl chain; R₁₁ and R₁₂ areeach independently H or a C₁ to C₄ linear or brarfched alkyl chain, onthe proviso that both R₁₁ and R₁₂ are not H; x is 1 to 4, and y is 1 to5, provided that x+y is 2 to 7; and z is 2 to
 5. 2. The cyclic peptideof claim 1 of the structural formula:

wherein R₃ and R₆ are as defined in claim
 1. 3. The cyclic peptide ofclaim 2 which is: Ac-cyc/o(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH;Ac-cyc/o(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH; or Ac-cyc/o(-Asp-Trp-D-Nal2-Arg-Nal 2-Lys)-OH.
 4. The cyclic peptide of claim 1 of the structuralformula:

wherein R₃ and R₆ are as defined in claim
 1. 5. The cyclic peptide ofclaim 4 which is: Ac-Nle-cyc/o(-Asp-His-D-Nal 2-Arg-Trp-Lys)-OH;Ac-Nle-cyc/o(-Asp-His-D-Nal 2-Arg-Nal 2-Lys)-OH; orAc-Nle-cyc/o(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-OH.
 6. The cyclic peptideof claim 1 of the structural formula:

wherein R₃, R₆, R₁₁ and R₁₂ are as defined in claim
 1. 7. The cyclicpeptide of claim 6 which is: Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal2-Lys)-NH—CH₂—CH₃; Ac-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂; orAc-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃.
 8. The cyclic peptideof claim 1 of the structural formula:

wherein R₃, R₅, R₆, R₁₁, R₁₂ and z are as defined in claim
 1. 9. Thecyclic peptide of claim 8 which is: H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal2-Lys)-NH—CH₂—CH₃; H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-NH—CH₃;H-cyclo(-Asp-Trp-D-Nal 2-Arg-Nal 2-Lys)-N(CH₃)₂; H-cyclo(-Asp-Trp-D-Nal2-Lys-Nal 2-Lys)-NH—CH₂—CH₃; H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal2-Lys)-NH—CH₃; or H-cyclo(-Asp-Trp-D-Nal 2-Lys-Nal 2-Lys)-N(CH₃)₂.
 10. Apharmaceutical preparation, comprising a cyclic peptide of claim 1 or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 11. A method of treating cachexia, comprisingadministering a pharmaceutically sufficient amount of a pharmaceuticalpreparation of claim 10 to a mammal, wherein the amount of thepharmaceutical preparation is effective to alleviate the symptom ofcachexia.
 12. A synthetic cyclic hexapeptide with a C-terminus hydroxylor N-alkyl group, wherein the N-alkyl group comprises one or two C₁ toC₄ linear or branched alkyl chains, the hexapeptide containing the coresequence His-D-Nal 2-X—Y or Trp-D-Nal 2-X—Y in positions 2 through 5,wherein X is an L-amino acid residue selected from the group consistingof Arg, Lys, Om, Harg and Hlys and Y is an L- or D-amino acid residueselected from the group consisting of Nal 1, Nal 2 Trp, wherein anyaromatic ring in the core sequence optionally includes one or two ringsubstituents, and when one or both ring substituents are present, arethe same or different and independently hydroxyl, halogen, alkyl, oraryl groups attached directly or through an ether linkage, and whereinthe cyclic hexapeptide is cyclized through the amino acid residue in the1 position and the amino acid residue in the 6 position by formation ofa covalent amide bond.
 13. The cyclic hexapeptide of claim 12 with anN-terminus Ac group.
 14. The cyclic hexapeptide of claim 12 wherein thehexapeptide is cyclized by formation of an amide bond between an aminogroup of a side chain of an amino acid in the 1 position or an aminogroup of the N-terminus group of the amino acid in the 1 position and aside chain carboxyl group of an amino acid residue at the 6 position.15. The cyclic hexapeptide of claim 12, wherein the hexapeptide iscyclized by formation of an amide bond between a side chain carboxylgroup of an amino acid residue in the 1 position and an amino group of aside chain of an amino acid at the 6 position.
 16. The cyclichexapeptide of claim 12 wherein the core sequence is His-D-Nal 2-X-Nal2.
 17. The cyclic hexapeptide of claim 12 wherein the core sequence isTrp-D-Nal 2-X-Nal
 2. 18. The cyclic hexapeptide of claim 12 wherein thecore sequence is His-D-Nal 2-X-Trp.